Helicase-Initiated Assembly of Macromolecular Machines Involved in DNA Replication
The research goal of this project is to obtain the first, comprehensive, physical model of the initial phase of the PriA helicase-directed formation of the primosome, a large protein-DNA complex, in E. coli, responsible for the restart of the DNA replication after the damage. The primosome, in its final state, comprises of as many as seven different proteins: PriA and DnaB helicases, DnaC, DnaG, DnaT, PriB, and PriC. Multiple and specific protein - protein, and protein - DNA interactions constitute integral part of this large molecular machine and dictate how this molecular motor assembles. However, the mechanistic details on how this intricate complex forms and accomplishes its function are unknown. In order to be able to put, such a complex molecular machine, together and understand, at the molecular level, its assembly process, discrete protein - protein and protein - DNA interactions of its components must be elucidated. Therefore, to begin with, the examination of the interactions of the PriA helicase, the first player in the assembly process, with various structures of the DNA, and the effect of the nucleotide cofactors on the interactions was addressed. The interactions of the PriB protein with PriA-DNA complex are considered to be the next, crucial, step in the assembly process of the primosome. In turn, the interactions of PriB with single stranded and double stranded DNA were examined. Only when such information was available, tertiary complexes could be studied. Finally, having elucidated the nature of PriA-ssDNA / -dsDNA and PriB-ssDNA / -dsDNA binary complexes, tertiary PriA-PriB-ssDNA, PriA-PriB-dsDNA, PriA-PriB-PAS complexes were examined. Consequently, direct quantitative model of two initial steps in the primosome assembly, involving the PriA and PriB proteins vis-à-vis the minimal primosome assembly site (PAS) of phage phiX174, was proposed. The studies presented here establish the first quantitative framework to study, at the molecular level, the assembly mechanism of the primosome, a paradigm of a large molecular machine involved in DNA replication. By building on the scaffold established in this research project, subsequent steps in the assembly process will be examined, to grant the first, comprehensive, molecular model of the primosome formation.